How to Retain Polar and Nonpolar Compounds on the Same HPLC Stationary Phase with an Isocratic Mobile Phase

Jun 01, 2006
Volume 2, Issue 9

Separation and retention of both polar and nonpolar compounds by the same stationary phase can be a useful approach for analyses of complex samples with a broad range of chemical properties. Typical stationary phases are designed for retention of either polar or nonpolar compounds so that multiple steps are required when designing a separation strategy. Hydride silica–based stationary phases are new materials with properties that allow for the simultaneous retention of both polar and nonpolar compounds over a range of aqueous–organic mobile phase compositions. Adjustment of the aqueous–organic ratio will determine whether polar or nonpolar compounds have greater retention.

High performance liquid chromatography (HPLC) stationary phases can be segregated by their ability to separate either polar on nonpolar compounds, that is, reversed-phase materials (C18, C8) strongly retain nonpolar solutes with polar solutes eluting at or near the void volume, and hydrophilic interaction chromatography (HILIC) and normal phase columns strongly retain polar analytes with nonpolar compounds being essentially nonretained (1). Increasingly, many analyses such as those encountered in drug discovery, proteomics, and metabolomics can be more complex with solutes encompassing a broad range of polarities. To overcome these deficiencies in column performance, more complex schemes of analysis might have to be devised to provide successfully qualitative and quantitative information about solutes with differing hydrophobicities and hydrophilicities. These approaches can include various types of sample preparation or two-dimensional chromatographic methods (2) as well as using chromatographic extremes of pH (3) and temperature (4). In most cases, such methodology can be cumbersome, time consuming, and damaging to your instrument and the HPLC column. In many instances, it would be desirable to have a stationary phase that can retain both polar and nonpolar compounds in an isocratic run so that a single separation strategy can be devised to analyze samples with a broad range of polarities.

Recently, a third type of chromatographic strategy has been developed on stationary phases utilizing silicon-hydride-based particles with a bonded phase (5–15). It is referred to as aqueous normal phase (ANP) chromatography. The principle of ANP chromatography is simple: retention behavior is analogous to that found in normal phase chromatography but the mobile phase has some water as part of the binary solvent. "Normal phase" implies that retention is greatest for polar solutes such as acids and bases. In addition, retention must increase as the amount of the nonpolar solvent in the mobile phase increases. So if the mobile phase consists of water and acetonitrile, retention increases as the amount of acetonitrile increases. Typically, in ANP chromatography, the amount of the nonpolar component in the mobile phase must be 60% or greater with the exact point of increased retention depending upon the solute and the organic component of the mobile phase.

If a stationary phase had the retention properties previously described and could only separate polar solutes, then it would be similar to a HILIC material (16–18). The term ANP is useful in distinguishing the hydride-based phases from typical HILIC phases. The hydride stationary phases also can retain nonpolar compounds by a traditional reversed-phase mechanism during the same isocratic run as described previously. Therefore, it is this dual retention capability that distinguishes the silicon-hydride material from other silica-based HPLC stationary phases. ANP chromatography is a useful term for indicating retention of ionizable–polar compounds on a silicon-hydride stationary phase also possessing reversed-phase capabilities — as opposed to a HILIC process that can separate only polar solutes.


Instrumentation: All chromatographic experiments utilized a model 1050 HPLC system with a diode-array detector (Agilent Technologies, Wilmington, Delaware) and interfaced to a MicroMass mass spectrometer (Waters Corp., Milford, Massachusetts). The Micromass Platform LC system was equipped with an Edwards model E2M30 rotary vacuum pump (Chell Instruments, Norfolk, UK) a Micromass Platform LC model M940150DC1 atmospheric pressure chemical ionization (APCI) probe, and a computer-based data acquisition system with MassLynx (version 3.4) software (Waters). The instrument was purged with high-pressure liquid nitrogen gas (100 psi). The acquisition parameters used for all the separations were as follows: APCI pin 3.20, cone 25, skimmer 2.0, source heater 140 °C, APCI probe temperature 600 °C, gas flow 250 mL/min. 0.5% formic acid was added to the mobile phase for ionizing the test samples. All liquid chromatography–mass spectrometry (LC–MS) separations were performed using the APCI probe in the positive ion mode.